This invention relates to synthesis of dithiols as reducing agents for disulfides.
The oxidation state of sulfhydryl groups influences the structure and activity of many biological systems; the thiol/disulfide interchange reaction is important in determining this state (Jocelyn, Biochemistry of the SH Group, Academic: London, 1972; and Gilbert, 63 Adv. Enzymol. 69, 1990). For example, thiol/disulfide interchange is important in the folding of proteins. Many enzymes require a cysteine in their active sites for catalysis: the thiol proteases (Glazer and Smith, The Enzymes Vol. III, 3rd ed., (Ed. P. D. Boyer) Academic: New York, 1971, pp. 501-546), enolase (Weiss et al., 109 J. Am. Chem. Soc. 7201, 1987), .beta.-ketoacylthiolase (Thompson et al., 28 Biochemistry 5735, 1989; and Mesamune et al., 61 Pure Appl. Chem. 303, 1989), and thioredoxin (Holmgren, 54 Annu. Rev. Biochem. 237, 1985) are rendered inactive by oxidative conversion of the reactive thiol group to a disulfide. Thiol/trisulfide interchange has been implicated as the "triggering event" in the cleavage of DNA by calichemicin and esperamicin. (Golik et al., 109 J. Am. Chem. Soc. 3461, 1987; Golik et al., 109 J. Am. Chem. Soc. 3462, 1987; Lee et al., 109 J. Am. Chem. Soc. 3464, 1987; and, Lee et al., 109 J. Am. Chem. Soc. 3466, 1987.)
In an effort to develop reagents useful in controlling thiol/disulfide interchange in aqueous solutions, we have previously examined a number of dithiols for their usefulness as reducing agents for disulfides. (Singh and Whitesides, 56 J. Org. Chem. 2332, 1991; and Lees et al., 56 J. Org. Chem. 7328, 1991.) Although several other reagents are already available for this reaction (Jocelyn, 143 Methods Enzymol. 246, 1987), there is still room for improvement. Dithiothreitol (DTT, Cleland's reagent) is one reagent that is widely used for reduction of a disulfide bond. (Cleland, 3 Biochemistry 480, 1964). It is a strong reductant, but expensive. It is also kinetically slow at pH=7. Mercaptoethanol (ME) is inexpensive, but it is a weak and slow reducing agent, and formation of mixed disulfides with ME is common.
We wished to design a reagent that would have properties superior to the compounds presently in use for the reduction of disulfide bonds. The practical properties that are important in the design of dithiols for the efficient reduction of acyclic disulfides are high solubility in aqueous solutions, low cost, low odor, and low toxicity. (Whitesides et al., 42 J. Org. Chem. 332, 1977). We were especially interested in the rate of reduction and the redox potential. In actual practice, rates of reduction that are 5-7 times faster than DTT have been observed (Lees et al., supra).
In addition to a fast rate of reduction, a useful dithiol should be strongly reducing in order to reduce disulfides quantitatively and to maintain thiols in the reduced state without the inconvenience of mixed disulfides. Since DTT is one of the most strongly reducing dithiols, the larger the equilibrium constant for thiol/disulfide exchange between a dithiol and DTT.sub.ox, the stronger the reductant. We have used this equilibrium to evaluate several dithiols in terms of their reduction strength.